Our work has focused on bringing retroviral gene transfer techniques developed in the Hematology Branch into clinical applications targeting human hematopoietic progenitor and stem cells. Over the past year we have enrolled a total of 13 patients undergoing autologous stem cell transplantation for aggressive treatment of breast cancer or multiple myeloma into clinical retroviral genetic marking trials. These trials were designed to answer important questions regarding the feasibility of using retroviral vectors to transfer genes into human hematopoietic stem and progenitor cells, the potential for using bone marrow versus peripheral blood stem and progenitor cells as targets for gene therapy, the pattern and durability of engraftment with bone marrow versus peripheral blood grafts after transplantation, and the source of relapse after autologous transplantation for these malignancies. 9/11 patients analyzed thus far post-transplant show evidence of hematopoietic cells containing the transfected gene. Two patients continue to show the presence of the marker gene at one year after transplantation. The level of marked cells is very low, and we are modifying the techniques used to get vector into the target stem cell to try and increase efficiency. No adverse events related to the gene transfer procedure have been observed. In preclinical in vitro and animal models we are exploring the use of serum-free transduction systems and concentrated, purified viral vectors to try and increase efficiency of gene transfer. We have shown that transduction can occur in a serum-free environment using highly purified vector preparations. We art also attempting to block cytokine inhibition of primitive cell cycling to allow better transduction. Based on the data we are generating in the clinical marking trials andents and murine and primate animal data, we are about to embark on two potentially therapeutic protocols: transfer of the multidrug-resistance gene to autologous marrow to confer chemoprotection in breast cancer patients, and transfer of the glucocerebrocidase gene to bone marrow or peripheral blood stem cells to treat Gaucher Disease. Both protocols are in the final regulatory review process. We have also collaborated on the extension of the human ADA gene therapy protocol to peripheral blood stem cells and cord blood cells in hopes of curing children with SCIDS with one gene-corrected cell infusion instead of multiple T-cell infusions. 3 newborns received transduced CD34+ cord blood cells, and show the transferred ADA gene in peripheral blood T cells over a year after transplantation. A new avenue of investigation in the murine model involves the use of primary marrow stromal (microenvironmental) cells as a target for gene therapy, as a vehicle to deliver stimulatory or inhibitory signals to the marrow. We have shown that the IL3 gene can be introduced into stromal cells, and these cells can engraft in the marrow and have a sustained biological effect cells, and these cells can engraft in the marrow and produce a sustained biological effect.